DESCRIPTION: (verbatim) Bioengineered skin shows great promise in the treatment of chronic wounds that are difficult to heal. Recently, in a prospective randomized trial of 293 patients, we have shown that graftskin, a bilayered skin construct (BSC) composed of neonatal keratinocytes and fibroblasts in type I bovine collagen, accelerates the healing of difficult to heal venous ulcers when compared to compression therapy alone. Yet, we don't know whether the allogeneic cells in BSC persist in the wound and how BSC accelerates healing. Much of the therapeutic effect of BSC is associated with stimulation of the wound's edges toward the center, but it is unclear whether this mechanism involves incorporation of donor cells into the wound margin. Recently, we have found that dermal fibroblasts cultured from the edge of venous ulcers are unresponsive to the action of transforming growth factor-beta-1 (TGF-beta-1), a critical peptide in the formation and deposition of collagen synthesis, and show decreased expression of TGF-beta receptors. The hypothesis of this proposal is that BSC is capable of stimulating the edges of venous ulcers to migrate and can restore the phenotypic make-up of resident wound cells at the margins of the wound. We propose the following specific aims: 1) Determine whether the edges of the wound are stimulated to migrate and whether, by immunohistochemisry and flow cytometry, cells at the edge become activated when BSC is placed only in the center of venous ulcers; 2) Determine by PCR and by fluorescence in situ hybridization whether BSC cells persist in venous ulcers at various times after its application; 3) Measure the response of BSC to injury by the use of flow cytometry, proliferative markers (i.e., ki67) and cytokine expression. 4) Determine whether BSC is capable of restoring responsiveness to TGF-beta-1 and expression of TGF-beta receptors in fibroblasts cultured from non-healing venous ulcers. The proposed studies will advance our understanding of how a bioengineered skin product works in a human chronic wound and provide insight into further bioengineering needs.